Displaying publications 1 - 20 of 219 in total

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  1. Immobilization of recombinant Escherichia coli on multi-walled carbon nanotubes for xylitol production
    Abd Rahman NH, Md Jahim J, Abdul Munaim MS, A Rahman R, Fuzi SFZ, Md Illias R
    Enzyme Microb Technol, 2020 Apr;135:109495.
    PMID: 32146929 DOI: 10.1016/j.enzmictec.2019.109495
    E. coli has been engineered to produce xylitol, but the production faces bottlenecks in terms of production yield and cell viability. In this study, recombinant E. coli (rE. coli) was immobilized on untreated and treated multiwalled carbon nanotubes (MWCNTs) for xylitol production. The immobilized rE. coli on untreated MWCNTs gave the highest xylitol production (5.47 g L-1) and a productivity of 0.22 g L-1 h-1. The doubling time for the immobilized cells increased up to 20.40 h and was higher than that of free cells (3.67 h). Cell lysis of the immobilized cells was reduced by up to 73 %, and plasmid stability improved by up to 17 % compared to those of free cells. Xylitol production using the optimum parameters (pH 7.4, 0.005 mM and 29 °C) achieved a xylitol production and productivity of 6.33 g L-1 and 0.26 g L-1 h-1, respectively. A seven-cycle repeated batch fermentation was carried out for up to 168 h, which showed maximum xylitol production of 7.36 g L-1 during the third cycle. Hence, this new adsorption immobilization system using MWCNTs is an alternative to improve the production of xylitol.
    Matched MeSH terms: Escherichia coli/genetics*
  2. Expression of recombinant human epidermal growth factor in Escherichia coli and characterization of its biological activity
    Abdull Razis AF, Ismail EN, Hambali Z, Abdullah MN, Ali AM, Mohd Lila MA
    Appl Biochem Biotechnol, 2008 Mar;144(3):249-61.
    PMID: 18556814
    Recombinant human epidermal growth factor (EGF) was successfully expressed as a fusion protein in Escherichia coli system. This system was used OmpA signal sequence to produce soluble protein into the periplasm of E. coli. Human EGF (hEGF) synthesized in bacterial cell was found to be similar in size with the original protein and molecular weight approximately at 6.8 kDa. Cell proliferation assay was conducted to characterize the biological activity of hEGF on human dermal fibroblasts. The synthesized hEGF was found to be functional as compared with authentic hEGF in stimulating cell proliferation and promoting growth of cell. In comparison of biological activity between synthesized and commercial hEGF on cell proliferation, the results showed there was no significant different. This finding indicates the synthesized hEGF in E. coli system is fully bioactive in vitro.
    Matched MeSH terms: Escherichia coli/genetics*
  3. Molecular characterization and phylogeny of Shiga toxin-producing E. coli (STEC) from imported beef meat in Malaysia
    Abuelhassan NN, Mutalib SA, Gimba FI, Yusoff WM
    Environ Sci Pollut Res Int, 2016 Sep;23(17):17553-62.
    PMID: 27234829 DOI: 10.1007/s11356-016-6954-0
    This study aimed at determining the presence and characterization of Escherichia coli and Shiga toxin-producing E. coli (STEC) from imported frozen beef meats. Seventy-four (74) frozen imported beef meat samples from two countries, India (42 samples) and Australia (32 samples), were collected and tested for E. coli. These samples were purchased from the frozen meat sections of five different supermarkets in different locations in Selangor, Malaysia, from April 2012 to October 2014. A total of 222 E. coli strains were isolated from the meat samples; 126 strains were isolated from country A (India), and 96 E. coli strains were from country of origin B (Australia), respectively. A total of 70 E. coli strains were identified and characterized. All E. coli strains were isolated into Fluorocult medium and identified using API 20E kit. All selected E. coli strains were characterized for Shiga toxin genes (stx1 and stx2). All biochemically identified E. coli in this study were further subjected to molecular detection through polymerase chain reaction (PCR) amplification and characterization using 16S ribosomal RNA (rRNA) gene of Shiga toxin-producing E. coli. Of the 70 E. coli strains, 11 strains were positive for both Shiga toxin genes (stx1 and stx2) and 11 (11/70) strains were positive for stx1 gene, while 25 (25/70) strains were positive for stx2 gene. The analysis of 16S rRNA gene of all the E. coli isolates in this study was successfully sequenced and analyzed, and based on sequence data obtained, a phylogenetic tree of the 16S rRNA gene was performed using Clustal W programme in MEGA 6.06 software. Phylogenetic tree showed that the E. coli isolates in our study cluster with the strain of E. coli isolated in other countries, which further confirm that the isolates of E. coli in this study are similar to those obtained in other studies. As a result, all the strains obtained in this study proved to be a strain of pathogenic E. coli, which may cause a serious outbreak of food-borne disease. The isolation of pathogenic E. coli strains from the imported meat samples calls for prudent management of imported meats by the relevant authorities.
    Matched MeSH terms: Shiga-Toxigenic Escherichia coli/genetics*
  4. Fulltext T4-like coliphage ΦKAZ14 virulent to pathogenic and extended spectrum β-lactamase-producing Escherichia coli of poultry origin
    Ahmad KA, Mohanmmed AS, Abas F, Chin SC
    Virol Sin, 2015 Feb;30(1):73-5.
    PMID: 25662886 DOI: 10.1007/s12250-014-3541-8
    Matched MeSH terms: Escherichia coli/genetics
  5. Inhibition of lysosomal vacuolar proton pump down-regulates cellular acidification and enhances E. coli bactofection efficiency
    Akinsola RO, Lee CW, Sim EUH, Narayanan K
    Anal Biochem, 2021 03 01;616:114088.
    PMID: 33358938 DOI: 10.1016/j.ab.2020.114088
    Endosomal escape is considered a crucial barrier that needs to be overcome by integrin-mediated E. coli for gene delivery into mammalian cells. Bafilomycin, a potent inhibitor of the H+ proton pump commonly employed to lower endosomal pH, was evaluated as part of the E. coli protocol during delivery. We found an increase in green fluorescent protein expression up 6.9, 3.2, 5.0, 2.8, and 4.5 fold in HeLa, HEK-293, A549, HT1080, and MCF-7 respectively, compared to untreated cells. Our result showed for the first time that Inhibition of lysosomal V-ATPase enhances E. coli efficiency.
    Matched MeSH terms: Escherichia coli/genetics
  6. Fulltext Phylogenetically Diverse Escherichia coli Strains from Chicken Coharbor Multiple Carbapenemase-Encoding Genes (bla NDM -bla OXA-blaIMP)
    Aklilu E, Harun A, Singh KKB, Ibrahim S, Kamaruzzaman NF
    Biomed Res Int, 2021;2021:5596502.
    PMID: 34660793 DOI: 10.1155/2021/5596502
    Carbapenem-resistant Enterobacteriaceae (CRE) has been a public health risk in several countries, and recent reports indicate the emergence of CRE in food animals. This study was conducted to investigate the occurrence, resistance patterns, and phylogenetic diversity of carbapenem-resistant E. coli (CREC) from chicken. Routine bacteriology, PCR detection of E. coli species, multiplex PCR to detect carbapenemase-encoding genes, and phylogeny of CRE E. coli were conducted. The results show that 24.36% (19/78) were identified as CREC based on the phenotypic identifications of which 17 were positive for the tested carbapenemases genes. The majority, 57.99% (11/19), of the isolates harbored multiple carbapenemase genes. Four isolates harbored all bla NDM, bla OXA, and bla IMP, and five and two different isolates harbored bla NDM and bla OXA and bla OXA and bla IMP, respectively. The meropenem, imipenem, and ertapenem MIC values for the isolates ranged from 2 μg/mL to ≥256 μg/mL. Phylogenetic grouping showed that the CREC isolates belonged to five different groups: groups A, B1, C, D, and unknown. The detection of CREC in this study shows that it has become an emerging problem in farm animals, particularly, in poultry farms. This also implies the potential public health risks posed by CRE from chicken to the consumers.
    Matched MeSH terms: Escherichia coli/genetics
  7. Cold-adapted RTX lipase from antarctic Pseudomonas sp. strain AMS8: isolation, molecular modeling and heterologous expression
    Ali MS, Ganasen M, Rahman RN, Chor AL, Salleh AB, Basri M
    Protein J, 2013 Apr;32(4):317-25.
    PMID: 23645400 DOI: 10.1007/s10930-013-9488-z
    A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfully isolated from strain AMS8, cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis revealed that lipAMS8 consist of 1,431 bp nucleotides that encoded a polypeptide consisting of 476 amino acids. It lacked an N-terminal signal peptide and contained a glycine- and aspartate-rich nonapeptide sequence at the C-terminus, which are known to be the characteristics of repeats-in-toxin bacterial lipases. Furthermore, the substrate binding site of lipAMS8 was identified as S(207), D(255) and H(313), based on homology modeling and multiple sequence alignment. Crude lipase exhibited maximum activity at 20 °C and retained almost 50 % of its activity at 10 °C. The molecular weight of lipAMS8 was estimated to be 50 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal expression level was attained using the recombinant plasmid pET32b/BL21(DE3) expressed at 15 °C for 8 h, induced by 0.1 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5.
    Matched MeSH terms: Escherichia coli/genetics
  8. Fulltext Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli
    Ali SA, Chew YW, Omar TC, Azman N
    PLoS One, 2015;10(12):e0144189.
    PMID: 26642325 DOI: 10.1371/journal.pone.0144189
    Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing β-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system.
    Matched MeSH terms: Escherichia coli/genetics*
  9. Fulltext FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium-Copy Number Plasmids in Escherichia coli
    Ali SA, Chew YW
    PLoS One, 2015;10(6):e0129547.
    PMID: 26057251 DOI: 10.1371/journal.pone.0129547
    Antibiotic resistance genes and antibiotics are frequently used to maintain plasmid vectors in bacterial hosts such as Escherichia coli. Due to the risk of spread of antibiotic resistance, the regulatory authorities discourage the use of antibiotic resistance genes/antibiotics for the maintenance of plasmid vectors in certain biotechnology applications. Overexpression of E. coli endogenous fabI gene and subsequent selection on Triclosan has been proposed as a practical alternative to traditional antibiotic selection systems. Unfortunately, overexpression of fabI cannot be used to select medium-copy number plasmids, typically used for the expression of heterologous proteins in E. coli. Here we report that Vibrio cholera FabV, a functional homologue of E. coli FabI, can be used as a suitable marker for the selection and maintenance of both high and medium-copy number plasmid vectors in E. coli.
    Matched MeSH terms: Escherichia coli/genetics*
  10. Recent Advances in Overexpression of Functional Recombinant Lipases
    Alias FL, Nezhad NG, Normi YM, Ali MSM, Budiman C, Leow TC
    Mol Biotechnol, 2023 Nov;65(11):1737-1749.
    PMID: 36971996 DOI: 10.1007/s12033-023-00725-y
    Heterologous functional expression of the recombinant lipases is typically a bottleneck due to the expression in the insoluble fraction as inclusion bodies (IBs) which are in inactive form. Due to the importance of lipases in various industrial applications, many investigations have been conducted to discover suitable approaches to obtain functional lipase or increase the expressed yield in the soluble fraction. The utilization of the appropriate prokaryotic and eukaryotic expression systems, along with the suitable vectors, promoters, and tags, has been recognized as a practical approach. One of the most powerful strategies to produce bioactive lipases is using the molecular chaperones co-expressed along with the target protein's genes into the expression host to produce the lipase in soluble fraction as a bioactive form. The refolding of expressed lipase from IBs (inactive) is another practical strategy which is usually carried out through chemical and physical methods. Based on recent investigations, the current review simultaneously highlights strategies to express the bioactive lipases and recover the bioactive lipases from the IBs in insoluble form.
    Matched MeSH terms: Escherichia coli/genetics
  11. Fulltext Risk factors and spatial distribution of extended spectrum β-lactamase-producing- Escherichia coli at retail poultry meat markets in Malaysia: a cross-sectional study
    Aliyu AB, Saleha AA, Jalila A, Zunita Z
    BMC Public Health, 2016 08 02;16:699.
    PMID: 27484086 DOI: 10.1186/s12889-016-3377-2
    BACKGROUND: The significant role of retail poultry meat as an important exposure pathway for the acquisition and transmission of extended spectrum β-lactamase-producing Escherichia coli (ESBL-EC) into the human population warrants understanding concerning those operational practices associated with dissemination of ESBL-EC in poultry meat retailing. Hence, the objective of this study was to determine the prevalence, spatial distribution and potential risk factors associated with the dissemination of ESBL-EC in poultry meat retail at wet-markets in Selangor, Malaysia.

    METHODS: Poultry meat (breast, wing, thigh, and keel) as well as the contact surfaces of weighing scales and cutting boards were sampled to detect ESBL-EC by using culture and disk combination methods and polymerase chain reaction assays. Besides, questionnaire was used to obtain data and information pertaining to those operational practices that may possibly explain the occurrence of ESBL-EC. The data were analysed using logistic regression analysis at 95 % CI.

    RESULTS: The overall prevalence of ESBL-EC was 48.8 % (95 % CI, 42 - 55 %). Among the risk factors that were explored, type of countertop, sanitation of the stall environment, source of cleaning water, and type of cutting board were found to be significantly associated with the presence of ESBL-EC.

    CONCLUSIONS: Thus, in order to prevent or reduce the presence of ESBL-EC and other contaminants at the retail-outlet, there is a need to design a process control system based on the current prevailing practices in order to reduce cross contamination, as well as to improve food safety and consumer health.

    Matched MeSH terms: Escherichia coli/genetics
  12. Fulltext Identification and real-time expression analysis of selected Toxoplasma gondii in-vivo induced antigens recognized by IgG and IgM in sera of acute toxoplasmosis patients
    Amerizadeh A, Khoo BY, Teh AY, Golkar M, Abdul Karim IZ, Osman S, et al.
    BMC Infect Dis, 2013;13:287.
    PMID: 23800344 DOI: 10.1186/1471-2334-13-287
    Toxoplasma gondii is an obligate intracellular zoonotic parasite of the phylum Apicomplexa which infects a wide range of warm-blooded animals, including humans. In this study in-vivo induced antigens of this parasite was investigated using in-vivo induced antigen technology (IVIAT) and pooled sera from patients with serological evidence of acute infection.
    Matched MeSH terms: Escherichia coli/genetics
  13. Biochemical Characterization of the Cytochrome P450 CYP107CB2 from Bacillus lehensis G1
    Ang SS, Salleh AB, Chor LT, Normi YM, Tejo BA, Rahman MBA, et al.
    Protein J, 2018 04;37(2):180-193.
    PMID: 29508210 DOI: 10.1007/s10930-018-9764-z
    The bioconversion of vitamin D3 catalyzed by cytochrome P450 (CYP) requires 25-hydroxylation and subsequent 1α-hydroxylation to produce the hormonal activated 1α,25-dihydroxyvitamin D3. Vitamin D3 25-hydroxylase catalyses the first step in the vitamin D3 biosynthetic pathway, essential in the de novo activation of vitamin D3. A CYP known as CYP107CB2 has been identified as a novel vitamin D hydroxylase in Bacillus lehensis G1. In order to deepen the understanding of this bacterial origin CYP107CB2, its detailed biological functions as well as biochemical characteristics were defined. CYP107CB2 was characterized through the absorption spectral analysis and accordingly, the enzyme was assayed for vitamin D3 hydroxylation activity. CYP-ligand characterization and catalysis optimization were conducted to increase the turnover of hydroxylated products in an NADPH-regenerating system. Results revealed that the over-expressed CYP107CB2 protein was dominantly cytosolic and the purified fraction showed a protein band at approximately 62 kDa on SDS-PAGE, indicative of CYP107CB2. Spectral analysis indicated that CYP107CB2 protein was properly folded and it was in the active form to catalyze vitamin D3 reaction at C25. HPLC and MS analysis from a reconstituted enzymatic reaction confirmed the hydroxylated products were 25-hydroxyitamin D3 and 1α,25-dihydroxyvitamin D3 when the substrates vitamin D3 and 1α-hydroxyvitamin D3 were used. Biochemical characterization shows that CYP107CB2 performed hydroxylation activity at 25 °C in pH 8 and successfully increased the production of 1α,25-dihydroxyvitamin D3 up to four fold. These findings show that CYP107CB2 has a biologically relevant vitamin D3 25-hydroxylase activity and further suggest the contribution of CYP family to the metabolism of vitamin D3.
    Matched MeSH terms: Escherichia coli/genetics
  14. An ultrasensitive hollow-silica-based biosensor for pathogenic Escherichia coli DNA detection
    Ariffin EY, Lee YH, Futra D, Tan LL, Karim NHA, Ibrahim NNN, et al.
    Anal Bioanal Chem, 2018 Mar;410(9):2363-2375.
    PMID: 29504083 DOI: 10.1007/s00216-018-0893-1
    A novel electrochemical DNA biosensor for ultrasensitive and selective quantitation of Escherichia coli DNA based on aminated hollow silica spheres (HSiSs) has been successfully developed. The HSiSs were synthesized with facile sonication and heating techniques. The HSiSs have an inner and an outer surface for DNA immobilization sites after they have been functionalized with 3-aminopropyltriethoxysilane. From field emission scanning electron microscopy images, the presence of pores was confirmed in the functionalized HSiSs. Furthermore, Brunauer-Emmett-Teller (BET) analysis indicated that the HSiSs have four times more surface area than silica spheres that have no pores. These aminated HSiSs were deposited onto a screen-printed carbon paste electrode containing a layer of gold nanoparticles (AuNPs) to form a AuNP/HSiS hybrid sensor membrane matrix. Aminated DNA probes were grafted onto the AuNP/HSiS-modified screen-printed electrode via imine covalent bonds with use of glutaraldehyde cross-linker. The DNA hybridization reaction was studied by differential pulse voltammetry using an anthraquinone redox intercalator as the electroactive DNA hybridization label. The DNA biosensor demonstrated a linear response over a wide target sequence concentration range of 1.0×10-12-1.0×10-2 μM, with a low detection limit of 8.17×10-14 μM (R2 = 0.99). The improved performance of the DNA biosensor appeared to be due to the hollow structure and rough surface morphology of the hollow silica particles, which greatly increased the total binding surface area for high DNA loading capacity. The HSiSs also facilitated molecule diffusion through the silica hollow structure, and substantially improved the overall DNA hybridization assay. Graphical abstract Step-by-step DNA biosensor fabrication based on aminated hollow silica spheres.
    Matched MeSH terms: Escherichia coli/genetics
  15. Gene profiling and characterization of arginine kinase-1 (MrAK-1) from freshwater giant prawn (Macrobrachium rosenbergii)
    Arockiaraj J, Vanaraja P, Easwvaran S, Singh A, Alinejaid T, Othman RY, et al.
    Fish Shellfish Immunol, 2011 Jul;31(1):81-9.
    PMID: 21549198 DOI: 10.1016/j.fsi.2011.04.004
    Arginine kinase-1 (MrAK-1) was sequenced from the freshwater prawn Macrobrachium rosenbergii using Illumina Solexa Genome Analyzer Technique. MrAK-1 consisted of 1068 bp nucleotide encoded 355 polypeptide with an estimated molecular mass of 40 kDa. MrAK-1 sequence contains a potential ATP:guanido phosphotransferases active domain site. The deduced amino acid sequence of MrAK-1 was compared with other 7 homologous arginine kinase (AK) and showed the highest identity (96%) with AK-1 from cherry shrimp Neocaridina denticulate. The qRT-PCR analysis revealed a broad expression of MrAK-1 with the highest expression in the muscle and the lowest in the eyestalk. The expression of MrAK-1 after challenge with the infectious hypodermal and hematopoietic necrosis virus (IHHNV) was tested in muscle. In addition, MrAK-1 was expressed in Escherichia coli by prokaryotic expression plasmid pMAL-c2x. The optimum temperature (30 °C) and pH (8.5) was determined for the enzyme activity assay. MrAK-1 showed significant (P < 0.05) activity towards 10-50 mM ATP concentration. The enzyme activity was inhibited by α-ketoglutarate, glucose and ATP at the concentration of 10, 50 and 100 mM respectively. Conclusively, the findings of this study indicated that MrAK-1 might play an important role in the coupling of energy production and utilization and the immune response in shrimps.
    Matched MeSH terms: Escherichia coli/genetics
  16. Molecular cloning, characterization and gene expression of an antioxidant enzyme catalase (MrCat) from Macrobrachium rosenbergii
    Arockiaraj J, Easwvaran S, Vanaraja P, Singh A, Othman RY, Bhassu S
    Fish Shellfish Immunol, 2012 May;32(5):670-82.
    PMID: 22293093 DOI: 10.1016/j.fsi.2012.01.013
    In this study, we reported a full length of catalase gene (designated as MrCat), identified from the transcriptome database of freshwater prawn Macrobrachium rosenbergii. The complete gene sequence of the MrCat is 2504 base pairs in length, and encodes 516 amino acids. The MrCat protein contains three domains such as catalase 1 (catalase proximal heme-ligand signature) at 350-358, catalase 2 (catalase proximal active site signature) at 60-76 and catalase 3 (catalase family profile) at 20-499. The mRNA expressions of MrCat in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) challenged M. rosenbergii were examined using quantitative real time polymerase chain reaction (qRT-PCR). The MrCat is highly expressed in digestive tract and all the other tissues (walking leg, gills, muscle, hemocyte, hepatopancreas, pleopods, brain and eye stalk) of M. rosenbergii taken for analysis. The expression is strongly up-regulated in digestive tract after IHHNV challenge. To understand its biological activity, the recombinant MrCat gene was constructed and expressed in Escherichia coli BL21 (DE3). The recombinant MrCat existed in high thermal stability and broad spectrum of pH, which showed over 95% enzyme activity between pH 5 and 10.5, and was stable from 40 °C to 70 °C, and exhibited 85-100% enzyme activity from 30 °C to 40 °C.
    Matched MeSH terms: Escherichia coli/genetics
  17. Fulltext Functional characterization of a new terpene synthase from Plectranthus amboinicus
    Ashaari NS, Ab Rahim MH, Sabri S, Lai KS, Song AA, Abdul Rahim R, et al.
    PLoS One, 2020;15(7):e0235416.
    PMID: 32614884 DOI: 10.1371/journal.pone.0235416
    Plectranthus amboinicus (Lour.) Spreng is an aromatic medicinal herb known for its therapeutic and nutritional properties attributed by the presence of monoterpene and sesquiterpene compounds. Up until now, research on terpenoid biosynthesis has focused on a few mint species with economic importance such as thyme and oregano, yet the terpene synthases responsible for monoterpene production in P. amboinicus have not been described. Here we report the isolation, heterologous expression and functional characterization of a terpene synthase involved in P. amboinicus terpenoid biosynthesis. A putative monoterpene synthase gene (PamTps1) from P. amboinicus was isolated with an open reading frame of 1797 bp encoding a predicted protein of 598 amino acids with molecular weight of 69.6 kDa. PamTps1 shares 60-70% amino acid sequence similarity with other known terpene synthases of Lamiaceae. The in vitro enzymatic activity of PamTps1 demonstrated the conversion of geranyl pyrophosphate and farnesyl pyrophosphate exclusively into linalool and nerolidol, respectively, and thus PamTps1 was classified as a linalool/nerolidol synthase. In vivo activity of PamTps1 in a recombinant Escherichia coli strain revealed production of linalool and nerolidol which correlated with its in vitro activity. This outcome validated the multi-substrate usage of this enzyme in producing linalool and nerolidol both in in vivo and in vitro systems. The transcript level of PamTps1 was prominent in the leaf during daytime as compared to the stem. Gas chromatography-mass spectrometry (GC-MS) and quantitative real-time PCR analyses showed that maximal linalool level was released during the daytime and lower at night following a diurnal circadian pattern which correlated with the PamTps1 expression pattern. The PamTps1 cloned herein provides a molecular basis for the terpenoid biosynthesis in this local herb that could be exploited for valuable production using metabolic engineering in both microbial and plant systems.
    Matched MeSH terms: Escherichia coli/genetics
  18. Application of the linear interaction energy method (LIE) to estimate the binding free energy values of Escherichia coli wild-type and mutant arginine repressor C-terminal domain (ArgRc)-l-arginine and ArgRc-l-citrulline protein-ligand complexes
    Asi AM, Rahman NA, Merican AF
    J Mol Graph Model, 2004 Mar;22(4):249-62.
    PMID: 15177077
    Protein-ligand binding free energy values of wild-type and mutant C-terminal domain of Escherichia coli arginine repressor (ArgRc) protein systems bound to L-arginine or L-citrulline molecules were calculated using the linear interaction energy (LIE) method by molecular dynamics (MD) simulation. The binding behaviour predicted by the dissociation constant (K(d)) calculations from the binding free energy values showed preferences for binding of L-arginine to the wild-type ArgRc but not to the mutant ArgRc(D128N). On the other hand, L-citrulline do not favour binding to wild-type ArgRc but prefer binding to mutant ArgRc(D128N). The dissociation constant for the wild-type ArgRc-L-arginine complex obtained in this study is in agreement with reported experimental results. Our results also support the experimental data for the binding of L-citrulline to the mutant ArgRc(D128N). These showed that LIE method for protein-ligand binding free energy calculation could be applied to the wild-type and the mutant E. coli ArgRc-L-arginine and ArgRc-L-citrulline protein-ligand complexes and possibly to other transcriptional repressor-co-repressor systems as well.
    Matched MeSH terms: Escherichia coli/genetics
  19. Identification of novel extracellular protein for PCB/biphenyl metabolism in Rhodococcus jostii RHA1
    Atago Y, Shimodaira J, Araki N, Bin Othman N, Zakaria Z, Fukuda M, et al.
    Biosci Biotechnol Biochem, 2016 May;80(5):1012-9.
    PMID: 26828632 DOI: 10.1080/09168451.2015.1127134
    Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.
    Matched MeSH terms: Escherichia coli/genetics
  20. Glyphosate-resistant goosegrass. Identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase
    Baerson SR, Rodriguez DJ, Tran M, Feng Y, Biest NA, Dill GM
    Plant Physiol, 2002 Jul;129(3):1265-75.
    PMID: 12114580
    The spontaneous occurrence of resistance to the herbicide glyphosate in weed species has been an extremely infrequent event, despite over 20 years of extensive use. Recently, a glyphosate-resistant biotype of goosegrass (Eleusine indica) was identified in Malaysia exhibiting an LD(50) value approximately 2- to 4-fold greater than the sensitive biotype collected from the same region. A comparison of the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity by glyphosate in extracts prepared from the resistant (R) and sensitive (S) biotypes revealed an approximately 5-fold higher IC(50)(glyphosate) for the (R) biotype. Sequence comparisons of the predicted EPSPS mature protein coding regions from both biotypes revealed four single-nucleotide differences, two of which result in amino acid changes. One of these changes, a proline to serine substitution at position 106 in the (R) biotype, corresponds to a substitution previously identified in a glyphosate-insensitive EPSPS enzyme from Salmonella typhimurium. Kinetic data generated for the recombinant enzymes suggests that the second substitution identified in the (R) EPSPS does not contribute significantly to its reduced glyphosate sensitivity. Escherichia coli aroA- (EPSPS deficient) strains expressing the mature EPSPS enzyme from the (R) biotype exhibited an approximately 3-fold increase in glyphosate tolerance relative to strains expressing the mature EPSPS from the (S) biotype. These results provide the first evidence for an altered EPSPS enzyme as an underlying component of evolved glyphosate resistance in any plant species.
    Matched MeSH terms: Escherichia coli/genetics
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